AXMI221z, AXMI222z, AXMI223z, AXMI224z and AXMI225z DELTA-ENDOTOXIN GENES AND METHODS FOR THEIR USE

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a toxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated toxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:21-32, or the nucleotide sequence set forth in SEQ ID NO: 1-20, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/030,415, filed Feb. 18, 2011, which claims the benefit of U.S.Provisional Application Ser. No. 61/305,802, filed Feb. 18, 2010, thecontents of which are herein incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“APA069US01DSEQLIST.txt”, created on Mar. 13, 2014, and having a size of132 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a Gram-positive spore forming soil bacteriumcharacterized by its ability to produce crystalline inclusions that arespecifically toxic to certain orders and species of insects, but areharmless to plants and other non-targeted organisms. For this reason,compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally-acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensishave potent insecticidal activity against predominantly Lepidopteran,Hemipteran, Dipteran, and Coleopteran larvae. These proteins also haveshown activity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga,and Acari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides, Marcel Dekker, Inc., New York, N.Y.) These proteins wereoriginally classified as CryI to CryV based primarily on theirinsecticidal activity. The major classes were Lepidoptera-specific (I),Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III),Diptera-specific (IV), and nematode-specific (V) and (VI). The proteinswere further classified into subfamilies; more highly related proteinswithin each family were assigned divisional letters such as Cry1A,Cry1B, Cry1C, etc. Even more closely related proteins within eachdivision were given names such as Cry1C1, Cry1C2, etc.

A new nomenclature was recently described for the Cry genes based uponamino acid sequence homology rather than insect target specificity(Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In thenew classification, each toxin is assigned a unique name incorporating aprimary rank (an Arabic number), a secondary rank (an uppercase letter),a tertiary rank (a lowercase letter), and a quaternary rank (anotherArabic number). In the new classification, Roman numerals have beenexchanged for Arabic numerals in the primary rank. Proteins with lessthan 45% sequence identity have different primary ranks, and thecriteria for secondary and tertiary ranks are 78% and 95%, respectively.

The crystal protein does not exhibit insecticidal activity until it hasbeen ingested and solubilized in the insect midgut. The ingestedprotoxin is hydrolyzed by proteases in the insect digestive tract to anactive toxic molecule. (Höfte and Whiteley (1989) Microbiol. Rev.53:242-255). This toxin binds to apical brush border receptors in themidgut of the target larvae and inserts into the apical membranecreating ion channels or pores, resulting in larval death.

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Because of the devastation that insects can confer, and the improvementin yield by controlling insect pests, there is a continual need todiscover new forms of pesticidal toxins.

SUMMARY OF INVENTION

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsectidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleotide sequences can be used in DNA constructs orexpression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise transformed bacteria, plants,plant cells, tissues, and seeds.

In particular, isolated nucleic acid molecules are provided that encodea pesticidal protein. Additionally, amino acid sequences correspondingto the pesticidal protein are encompassed. In particular, the presentinvention provides for an isolated nucleic acid molecule comprising anucleotide sequence encoding the amino acid sequence shown in SEQ IDNO:21-32 or a nucleotide sequence set forth in SEQ ID NO: 1-5, as wellas variants and fragments thereof. Nucleotide sequences that arecomplementary to a nucleotide sequence of the invention, or thathybridize to a sequence of the invention are also encompassed. Syntheticnucleotide sequences encoding the polypeptides disclosed herein are alsoset forth in SEQ ID NO:6-20.

Methods are provided for producing the polypeptides of the invention,and for using those polypeptides for controlling or killing alepidopteran, hemipteran, coleopteran, nematode, or dipteran pest.Methods and kits for detecting the nucleic acids and polypeptides of theinvention in a sample are also included.

The compositions and methods of the invention are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the invention are alsouseful for generating altered or improved proteins that have pesticidalactivity, or for detecting the presence of pesticidal proteins ornucleic acids in products or organisms.

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating pest resistance or tolerance in organisms, particularlyplants or plant cells. By “resistance” is intended that the pest (e.g.,insect) is killed upon ingestion or other contact with the polypeptidesof the invention. By “tolerance” is intended an impairment or reductionin the movement, feeding, reproduction, or other functions of the pest.The methods involve transforming organisms with a nucleotide sequenceencoding a pesticidal protein of the invention. In particular, thenucleotide sequences of the invention are useful for preparing plantsand microorganisms that possess pesticidal activity. Thus, transformedbacteria, plants, plant cells, plant tissues and seeds are provided.Compositions are pesticidal nucleic acids and proteins of Bacillus orother species. The sequences find use in the construction of expressionvectors for subsequent transformation into organisms of interest, asprobes for the isolation of other homologous (or partially homologous)genes, and for the generation of altered pesticidal proteins by methodsknown in the art, such as domain swapping or DNA shuffling, for example,with members of the Cry1, Cry2, and Cry9 families of endotoxins. Theproteins find use in controlling or killing lepidopteran, hemipteran,coleopteran, dipteran, and nematode pest populations and for producingcompositions with pesticidal activity.

By “pesticidal toxin” or “pesticidal protein” is intended a toxin thathas toxic activity against one or more pests, including, but not limitedto, members of the Lepidoptera, Diptera, and Coleoptera orders, or theNematoda phylum, or a protein that has homology to such a protein.Pesticidal proteins have been isolated from organisms including, forexample, Bacillus sp., Clostridium bifermentans and Paenibacilluspopilliae. Pesticidal proteins include amino acid sequences deduced fromthe full-length nucleotide sequences disclosed herein, and amino acidsequences that are shorter than the full-length sequences, either due tothe use of an alternate downstream start site, or due to processing thatproduces a shorter protein having pesticidal activity. Processing mayoccur in the organism the protein is expressed in, or in the pest afteringestion of the protein.

Pesticidal proteins encompass delta-endotoxins. Delta-endotoxins includeproteins identified as cry1 through cry43, cyt1 and cyt2, and Cyt-liketoxin. There are currently over 250 known species of delta-endotoxinswith a wide range of specificities and toxicities. For an expansive listsee Crickmore et al. (1998), Microbiol. Mol. Biol. Rev. 62:807-813, andfor regular updates see Crickmore et al. (2003) “Bacillus thuringiensistoxin nomenclature,” on the worldwide web atbiols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.

Thus, provided herein are novel isolated nucleotide sequences thatconfer pesticidal activity. These isolated nucleotide sequences encodepolypeptides with homology to known delta-endotoxins or binary toxins.Also provided are the amino acid sequences of the pesticidal proteins.The protein resulting from translation of this gene allows cells tocontrol or kill pests that ingest it.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding pesticidalproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules encoding proteins with regionsof sequence homology. As used herein, the term “nucleic acid molecule”is intended to include DNA molecules (e.g., recombinant DNA, cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” or “recombinant” nucleic acid sequence (or DNA) is usedherein to refer to a nucleic acid sequence (or DNA) that is no longer inits natural environment, for example in an in vitro or in a recombinantbacterial or plant host cell. In some embodiments, an isolated orrecombinant nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forpurposes of the invention, “isolated” when used to refer to nucleic acidmolecules excludes isolated chromosomes. For example, in variousembodiments, the isolated delta-endotoxin encoding nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1kb of nucleotide sequences that naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. In various embodiments, a delta-endotoxin protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofnon-delta-endotoxin protein (also referred to herein as a “contaminatingprotein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO: 1-20, and variants,fragments, and complements thereof. By “complement” is intended anucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. The corresponding amino acidsequence for the pesticidal protein encoded by this nucleotide sequenceare set forth in SEQ ID NO:21-32.

Nucleic acid molecules that are fragments of these nucleotide sequencesencoding pesticidal proteins are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a pesticidal protein. A fragment of a nucleotidesequence may encode a biologically active portion of a pesticidalprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. Nucleic acidmolecules that are fragments of a nucleotide sequence encoding apesticidal protein comprise at least about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400 contiguousnucleotides, or up to the number of nucleotides present in a full-lengthnucleotide sequence encoding a pesticidal protein disclosed herein,depending upon the intended use. By “contiguous” nucleotides is intendednucleotide residues that are immediately adjacent to one another.Fragments of the nucleotide sequences of the present invention willencode protein fragments that retain the biological activity of thepesticidal protein and, hence, retain pesticidal activity. By “retainsactivity” is intended that the fragment will have at least about 30%, atleast about 50%, at least about 70%, 80%, 90%, 95% or higher of thepesticidal activity of the pesticidal protein. In one embodiment, thepesticidal activity is coleoptericidal activity. In another embodiment,the pesticidal activity is lepidoptericidal activity. In anotherembodiment, the pesticidal activity is nematocidal activity. In anotherembodiment, the pesticidal activity is diptericidal activity. In anotherembodiment, the pesticidal activity is hemiptericidal activity. Methodsfor measuring pesticidal activity are well known in the art. See, forexample, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrewset al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. ofEconomic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all ofwhich are herein incorporated by reference in their entirety.

A fragment of a nucleotide sequence encoding a pesticidal protein thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450 contiguous amino acids, or up to the total number ofamino acids present in a full-length pesticidal protein of theinvention. In some embodiments, the fragment is a proteolytic cleavagefragment. For example, the proteolytic cleavage fragment may have anN-terminal or a C-terminal truncation of at least about 100 amino acids,about 120, about 130, about 140, about 150, or about 160 amino acidsrelative to SEQ ID NO:21-32. In some embodiments, the fragmentsencompassed herein result from the removal of the C-terminalcrystallization domain, e.g., by proteolysis or by insertion of a stopcodon in the coding sequence. See, for example, the truncated amino acidsequences set forth in SEQ ID NO:22, 23, 25, 26, and 32. It will beunderstood that the truncation site may vary by 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or more amino acids on either side of thetruncation site represented by the terminus of SEQ ID NO:22, 23, 25, 26,and 32 (compared to the corresponding full-length sequence).

Preferred pesticidal proteins of the present invention are encoded by anucleotide sequence sufficiently identical to the nucleotide sequence ofSEQ ID NO: 1-20. By “sufficiently identical” is intended an amino acidor nucleotide sequence that has at least about 60% or 65% sequenceidentity, about 70% or 75% sequence identity, about 80% or 85% sequenceidentity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the percent identity iscalculated across the entirety of the reference sequence (i.e., thesequence disclosed herein as any of SEQ ID NO: 1-20). The percentidentity between two sequences can be determined using techniquessimilar to those described below, with or without allowing gaps. Incalculating percent identity, typically exact matches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous topesticidal-like nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to pesticidalprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48 (3):443-453, will be usedto determine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10. The invention alsoencompasses variant nucleic acid molecules. “Variants” of the pesticidalprotein encoding nucleotide sequences include those sequences thatencode the pesticidal proteins disclosed herein but that differconservatively because of the degeneracy of the genetic code as well asthose that are sufficiently identical as discussed above. Naturallyoccurring allelic variants can be identified with the use of well-knownmolecular biology techniques, such as polymerase chain reaction (PCR)and hybridization techniques as outlined below. Variant nucleotidesequences also include synthetically derived nucleotide sequences thathave been generated, for example, by using site-directed mutagenesis butwhich still encode the pesticidal proteins disclosed in the presentinvention as discussed below. Variant proteins encompassed by thepresent invention are biologically active, that is they continue topossess the desired biological activity of the native protein, that is,pesticidal activity. By “retains activity” is intended that the variantwill have at least about 30%, at least about 50%, at least about 70%, orat least about 80% of the pesticidal activity of the native protein.Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al.(1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedpesticidal proteins, without altering the biological activity of theproteins. Thus, variant isolated nucleic acid molecules can be createdby introducing one or more nucleotide substitutions, additions, ordeletions into the corresponding nucleotide sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a pesticidal protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the invention (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theinvention (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingpesticidal sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and Innis, et al. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, NY).

In a hybridization method, all or part of the pesticidal nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known pesticidalprotein-encoding nucleotide sequence disclosed herein. Degenerateprimers designed on the basis of conserved nucleotides or amino acidresidues in the nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, at least about 25, at least about 50, 75, 100, 125, 150,175, or 200 consecutive nucleotides of nucleotide sequence encoding apesticidal protein of the invention or a fragment or variant thereof.Methods for the preparation of probes for hybridization are generallyknown in the art and are disclosed in Sambrook and Russell, 2001, supraherein incorporated by reference.

For example, an entire pesticidal protein sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding pesticidal protein-likesequences and messenger RNAs. To achieve specific hybridization under avariety of conditions, such probes include sequences that are unique andare preferably at least about 10 nucleotides in length, or at leastabout 20 nucleotides in length. Such probes may be used to amplifycorresponding pesticidal sequences from a chosen organism by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Pesticidal proteins are also encompassed within the present invention.By “pesticidal protein” is intended a protein having the amino acidsequence set forth in SEQ ID NO:21-32. Fragments, biologically activeportions, and variants thereof are also provided, and may be used topractice the methods of the present invention. An “isolated protein” isused to refer to a protein that is no longer in its natural environment,for example in vitro or in a recombinant bacterial or plant host cell.An “isolated protein” or a “recombinant protein” is used to refer to aprotein that is no longer in its natural environment, for example invitro or in a recombinant bacterial or plant host cell.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO:21-32, and that exhibitpesticidal activity. A biologically active portion of a pesticidalprotein can be a polypeptide that is, for example, 10, 25, 50, 100, 150,200, 250 or more amino acids in length. Such biologically activeportions can be prepared by recombinant techniques and evaluated forpesticidal activity. Methods for measuring pesticidal activity are wellknown in the art. See, for example, Czapla and Lang (1990) J. Econ.Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206;Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S.Pat. No. 5,743,477, all of which are herein incorporated by reference intheir entirety. As used here, a fragment comprises at least 8 contiguousamino acids of SEQ ID NO:21-32. The invention encompasses otherfragments, however, such as any fragment in the protein greater thanabout 10, 20, 30, 50, 100, 150, 200, 250, or 300 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of any of SEQ ID NO:21-32. Variants alsoinclude polypeptides encoded by a nucleic acid molecule that hybridizesto the nucleic acid molecule of SEQ ID NO: 1-20, or a complementthereof, under stringent conditions. Variants include polypeptides thatdiffer in amino acid sequence due to mutagenesis. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, retaining pesticidal activity. In some embodiments,the variants have improved activity relative to the native protein.Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485;Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J.of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all ofwhich are herein incorporated by reference in their entirety.

Bacterial genes, such as the axmi genes of this invention, quite oftenpossess multiple methionine initiation codons in proximity to the startof the open reading frame. Often, translation initiation at one or moreof these start codons will lead to generation of a functional protein.These start codons can include ATG codons. However, bacteria such asBacillus sp. also recognize the codon GTG as a start codon, and proteinsthat initiate translation at GTG codons contain a methionine at thefirst amino acid. On rare occasions, translation in bacterial systemscan initiate at a TTG codon, though in this event the TTG encodes amethionine. Furthermore, it is not often determined a priori which ofthese codons are used naturally in the bacterium. Thus, it is understoodthat use of one of the alternate methionine codons may also lead togeneration of pesticidal proteins. See, for example, the alternate startsite for the AXMI223z protein set forth in SEQ ID NO: 28 and thealternate start site for AXMI224z protein set forth in SEQ ID NO:30.These pesticidal proteins are encompassed in the present invention andmay be used in the methods of the present invention. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a pesticidal protein may bealtered by various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by a pesticidal protein of the present invention. Thisprotein may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions of one or moreamino acids of SEQ ID NO:21-32, including up to about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 15,about 20, about 25, about 30, about 35, about 40, about 45, about 50,about 55, about 60, about 65, about 70, about 75, about 80, about 85,about 90, about 100, about 105, about 110, about 115, about 120, about125, about 130, about 135, about 140, about 145, about 150, about 155,or more amino acid substitutions, deletions or insertions. Methods forsuch manipulations are generally known in the art. For example, aminoacid sequence variants of a pesticidal protein can be prepared bymutations in the DNA. This may also be accomplished by one of severalforms of mutagenesis and/or in directed evolution. In some aspects, thechanges encoded in the amino acid sequence will not substantially affectthe function of the protein. Such variants will possess the desiredpesticidal activity. However, it is understood that the ability of apesticidal protein to confer pesticidal activity may be improved by theuse of such techniques upon the compositions of this invention. Forexample, one may express a pesticidal protein in host cells that exhibithigh rates of base misincorporation during DNA replication, such as XL-1Red (Stratagene, La Jolla, Calif.). After propagation in such strains,one can isolate the DNA (for example by preparing plasmid DNA, or byamplifying by PCR and cloning the resulting PCR fragment into a vector),culture the pesticidal protein mutations in a non-mutagenic strain, andidentify mutated genes with pesticidal activity, for example byperforming an assay to test for pesticidal activity. Generally, theprotein is mixed and used in feeding assays. See, for example Marrone etal. (1985) J. of Economic Entomology 78:290-293. Such assays can includecontacting plants with one or more pests and determining the plant'sability to survive and/or cause the death of the pests. Examples ofmutations that result in increased toxicity are found in Schnepf et al.(1998) Microbiol. Mol. Biol. Rev. 62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent pesticidal protein coding regions can be used to create a newpesticidal protein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneof the invention and other known pesticidal genes to obtain a new genecoding for a protein with an improved property of interest, such as anincreased insecticidal activity. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredpesticidal proteins. Domains may be swapped between pesticidal proteins,resulting in hybrid or chimeric toxins with improved pesticidal activityor target spectrum. Methods for generating recombinant proteins andtesting them for pesticidal activity are well known in the art (see, forexample, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; deMaagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al.(1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol.Chem. 265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol.65:2918-2925).

Vectors

A pesticidal sequence of the invention may be provided in an expressioncassette for expression in a plant of interest. By “plant expressioncassette” is intended a DNA construct that is capable of resulting inthe expression of a protein from an open reading frame in a plant cell.Typically these contain a promoter and a coding sequence. Often, suchconstructs will also contain a 3′ untranslated region. Such constructsmay contain a “signal sequence” or “leader sequence” to facilitateco-translational or post-translational transport of the peptide tocertain intracellular structures such as the chloroplast (or otherplastid), endoplasmic reticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.Insecticidal toxins of bacteria are often synthesized as protoxins,which are protolytically activated in the gut of the target pest (Chang(1987) Methods Enzymol. 153:507-516). In some embodiments of the presentinvention, the signal sequence is located in the native sequence, or maybe derived from a sequence of the invention. By “leader sequence” isintended any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

“Promoter” refers to a nucleic acid sequence that functions to directtranscription of a downstream coding sequence. The promoter togetherwith other transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the pesticidal sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

In one embodiment, the pesticidal protein is targeted to the chloroplastfor expression. In this manner, where the pesticidal protein is notdirectly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe pesticidal protein to the chloroplasts. Such transit peptides areknown in the art. See, for example, Von Heijne et al. (1991) Plant Mol.Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; andShah et al. (1986) Science 233:478-481.

The pesticidal gene to be targeted to the chloroplast may be optimizedfor expression in the chloroplast to account for differences in codonusage between the plant nucleus and this organelle. In this manner, thenucleic acids of interest may be synthesized using chloroplast-preferredcodons. See, for example, U.S. Pat. No. 5,380,831, herein incorporatedby reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell. “Heterologous” generally refers to the nucleic acid sequences thatare not endogenous to the cell or part of the native genome in whichthey are present, and have been added to the cell by infection,transfection, microinjection, electroporation, microprojection, or thelike.

The transgenic plants of the invention express one or more of the noveltoxin sequences disclosed herein. In various embodiments, the transgenicplant further comprises one or more additional genes for insectresistance (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C,Cry1D, Cry1E, and Cry1F families; Cry2, such as members of the Cry2Afamily; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E,and Cry9F families; etc.). It will be understood by one of skill in theart that the transgenic plant may comprise any gene imparting anagronomic trait of interest.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The pesticidal gene of the invention may bemodified to obtain or enhance expression in plant cells. Typically aconstruct that expresses such a protein would contain a promoter todrive transcription of the gene, as well as a 3′ untranslated region toallow transcription termination and polyadenylation. The organization ofsuch constructs is well known in the art. In some instances, it may beuseful to engineer the gene such that the resulting peptide is secreted,or otherwise targeted within the plant cell. For example, the gene canbe engineered to contain a signal peptide to facilitate transfer of thepeptide to the endoplasmic reticulum. It may also be preferable toengineer the plant expression cassette to contain an intron, such thatmRNA processing of the intron is required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors”. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the pesticidal gene are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thepesticidal protein.

Pesticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a pesticidal protein that has pesticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, biolistic transformation,and non-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a pesticidal protein may be isolated bycommon methods described in the art, for example by transformation ofcallus, selection of transformed callus, and regeneration of fertileplants from such transgenic callus. In such process, one may use anygene as a selectable marker so long as its expression in plant cellsconfers ability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for pesticidal activity.

Fertile plants expressing a pesticidal protein may be tested forpesticidal activity, and the plants showing optimal activity selectedfor further breeding. Methods are available in the art to assay for pestactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

Use in Pesticidal Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in pesticide control orin engineering other organisms as pesticidal agents are known in theart. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain a pesticidal gene and protein may be usedfor protecting agricultural crops and products from pests. In one aspectof the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

Alternatively, the pesticide is produced by introducing a pesticidalgene into a cellular host. Expression of the pesticidal gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. In one aspect of this invention, these cells are thentreated under conditions that prolong the activity of the toxin producedin the cell when the cell is applied to the environment of targetpest(s). The resulting product retains the toxicity of the toxin. Thesenaturally encapsulated pesticides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein. Alternatively,one may formulate the cells expressing a gene of this invention such asto allow application of the resulting material as a pesticide.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, pesticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, hemipteran, dipteran, or coleopteran pests may be killedor reduced in numbers in a given area by the methods of the invention,or may be prophylactically applied to an environmental area to preventinfestation by a susceptible pest. Preferably the pest ingests, or iscontacted with, a pesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrimidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylimidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachimidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,and Tineidae.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Siphaflava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which saidpolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a lepidopteran, coleopteran,dipteran, hemipteran, or nematode pest, and said field is infested witha lepidopteran, hemipteran, coleopteran, dipteran, or nematode pest. Asdefined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. By “biomass” is intended anymeasured plant product. An increase in biomass production is anyimprovement in the yield of the measured plant product. Increasing plantyield has several commercial applications. For example, increasing plantleaf biomass may increase the yield of leafy vegetables for human oranimal consumption. Additionally, increasing leaf biomass can be used toincrease production of plant-derived pharmaceutical or industrialproducts. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

The plants can also be treated with one or more chemical compositions,including one or more herbicide, insecticides, or fungicides. Exemplarychemical compositions include: Fruits/Vegetables Herbicides: Atrazine,Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine,Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat,Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis,Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin,Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate,Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran,Fluacrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen,Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr,Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide,Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen,Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb,Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Fozthiazate,Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox,Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron,Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam,Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim,Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph,Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid,Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione,Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron,Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet,Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin,Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos,Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron,Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin,Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos,Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin,Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan,Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides:Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron,Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron,Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet,Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid,Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor,Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; RiceInsecticides: Diazinon, Fenitrothion, Fenobucarb, Monocrotophos,Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid,Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran,Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin,Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos,Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:

Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLES Example 1 Discovery of Novel Pesticidal Genes fromBacillus thuringiensis

Novel pesticidal genes were identified from bacterial strain Zj22 usingthe following steps:

-   -   Preparation of extrachromosomal DNA from the strain.        Extrachromosomal DNA contains a mixture of some or all of the        following: plasmids of various size; phage chromosomes; genomic        DNA fragments not separated by the purification protocol; other        uncharacterized extrachromosomal molecules.    -   Mechanical or enzymatic shearing of the extrachromosomal DNA to        generate size-distributed fragments.    -   Sequencing of the fragmented DNA by high-throughput        pyrosequencing methods.    -   Identification of putative toxin genes via homology and/or other        computational analyses.    -   When required, sequence finishing of the gene of interest by one        of several PCR or cloning strategies (e.g. TAIL-PCR).

TABLE 1 Novel genes identified from strain Zj22 Molecular NucleotideGene weight SEQ ID Amino acid name (kD) Closest homolog NO SEQ ID NOAxmi221z 138.3 62.1% Cry9Aa 1 21 60.1% Cry1Aa 22 (truncated) 84.0%Cry9Aa 23 (truncated) (truncated) Axmi222z 141.1 86.5% Cry1Bf 2 24 86.4%Cry1Ba 25 (truncated) 76.2% Cry1Bf 26 (truncated) (truncated) 76.2%Cry1Ba (truncated) Axmi223z 80.9 84.7% Cry1Ia 3 27 82.1% Cry1If 28(alternate 81.9% Cry1Id start site) 81.8% Cry1Ie 81.2% Cry1Ib 78.2%Cry1Ic Axmi224z 75 98.9% Cry2Af 4 29 93.5% Cry2Ab1 30 (alternate 93.0%Cry2Ae1 start site) 91.2% Cry2Ad1 Axmi225z 133.2 98.6% Cry1Ab18 5 3194.8% Axmi112 32 (truncated) 94.8% Cry1Ae1 94.8% Cry1Ab1 98.6% Cry1Ab18(truncated) 98.0% Cry1Ab1 (truncated) 95.6% Cry1Ae1 (truncated) 95.4%Axmi112 (truncated)

The toxin gene disclosed herein is amplified by PCR from pAX980, and thePCR product is cloned into the Bacillus expression vector pAX916, oranother suitable vector, by methods well known in the art. The resultingBacillus strain, containing the vector with axmi gene is cultured on aconventional growth media, such as CYS media (10 g/l Bacto-casitone; 3g/l yeast extract; 6 g/l KH₂PO₄; 14 g/l K₂HPO₄; 0.5 mM MgSO₄; 0.05 mMMnCl₂; 0.05 mM FeSO₄), until sporulation is evident by microscopicexamination. Samples are prepared and tested for activity in bioassays.

Example 2 Assays for Pesticidal Activity

The nucleotide sequences of the invention can be tested for theirability to produce pesticidal proteins. The ability of a pesticidalprotein to act as a pesticide upon a pest is often assessed in a numberof ways. One way well known in the art is to perform a feeding assay. Insuch a feeding assay, one exposes the pest to a sample containing eithercompounds to be tested or control samples. Often this is performed byplacing the material to be tested, or a suitable dilution of suchmaterial, onto a material that the pest will ingest, such as anartificial diet. The material to be tested may be composed of a liquid,solid, or slurry. The material to be tested may be placed upon thesurface and then allowed to dry. Alternatively, the material to betested may be mixed with a molten artificial diet, then dispensed intothe assay chamber. The assay chamber may be, for example, a cup, a dish,or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson and Preisler, eds. (1992)Pesticide bioassays with arthropods, CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals ArthropodManagement Tests and Journal of Economic Entomology or by discussionwith members of the Entomological Society of America (ESA).

In some embodiments, the DNA regions encoding the toxin domains ofdelta-endotoxins disclosed herein are cloned into the E. coli expressionvector pMAL-C4x behind the malE gene coding for Maltose binding protein(MBP). These in-frame fusions result in MBP-Axmi fusion proteinsexpression in E. coli.

For expression in E. coli, BL21*DE3 are transformed with individualplasmids. Single colonies are inoculated in LB supplemented withcarbenicillin and glucose, and grown overnight at 37° C. The followingday, fresh medium is inoculated with 1% of overnight culture and grownat 37° C. to logarithmic phase. Subsequently, cultures are induced with0.3 mM IPTG overnight at 20° C. Each cell pellet is suspended in 20 mMTris-C1 buffer, pH 7.4+200 mM NaCl+1 mM DTT+protease inhibitors andsonicated. Analysis by SDS-PAGE can be used to confirm expression of thefusion proteins.

Total cell free extracts are then run over amylose column attached tofast protein liquid chromatography (FPLC) for affinity purification ofMBP-axmi fusion proteins. Bound fusion proteins are eluted from theresin with 10 mM maltose solution. Purified fusion proteins are thencleaved with either Factor Xa or trypsin to remove the amino terminalMBP tag from the Axmi protein. Cleavage and solubility of the proteinscan be determined by SDS-PAGE

Example 3 Expression and Purification of Axmiz Genes

Truncated versions of axmi221z and axmi222z were cloned into themaltose-binding protein (MBP) expression vector, resulting in pAX5092and pAX5093, respectively. Expression of the resulting fusion proteinwas induced by IPTG. Protein was then purified through a maltose columnand cleaved with protease Factor Xa to generate the untagged, purifiedprotein. The truncated 6-his axmi221z and axmi222z proteins were alsopurified on a cobalt column and submitted for bioassays.

Full-length and truncated versions of some genes were cloned into vectorpRSF-1b as shown in Table 2. By virtue of cloning into this vector, theresulting expressed protein contains an additional six N-terminalhistidine residues.

The DNA regions encoding the toxin domains of some genes were separatelycloned into an E. coli expression vector pMAL-C4x behind the malE genecoding for Maltose binding protein (MBP) as shown in Table 2. Thesein-frame fusions resulted in MBP-AXMI fusion proteins expression in E.coli. Each of the proteins produced from the constructs above weretested in bioassays as a 10× concentrated pellet.

TABLE 2 Axmiz constructs SEQ ID NO: of protein construct encoded by genename backbone vector construct Axmi221z (full length) pAX5095 pRSF-1b 21Axmi221z (full length) pAX7611 pAX916 21 Axmi221z (trun2) pAX5092pMAL-C4x 23 Axmi221z (trun2) pAX5094 pRSF-1b 23 Axmi221z (trun2) pAX7610pRSF-1b 23 Axmi222z (full length) pAX5097 pRSF-1b 24 Axmi222z (fulllength) pAX7613 pAX916 24 Axmi222z (trun2) pAX5093 pMAL-C4x 26 Axmi222z(trun2) pAX5096 pRSF-1b 26 Axmi222z (trun2) pAX7612 pAX916 26 Axmi223z(full length) pAX6887 pMAL-C4x 27 Axmi223z pAX6888 pMAL-C4x 28(alternate start site) Axmi224z pAX7634 pRSF-1b 30 (alternate startsite) Axmi224z pAX6890 pMAL-C4x 30 (alternate start site) Axmi225z(trun) pAX6891 pMAL-C4x 32

For expression of protein in E. coli, BL21*DE3 was transformed withindividual plasmids. A single colony was inoculated into LB mediasupplemented with carbenicillin and glucose, and grown overnight at 37°C. The following day, fresh medium was inoculated with 1% of overnightculture and grown at 37° C. to logarithmic phase. Subsequently, cultureswere induced with 0.3 mM IPTG overnight at 20° C. Each cell pellet wassuspended in 20 mM Tris-Cl buffer, pH 7.4+200 mM NaCl+1 mM DTT+proteaseinhibitors and sonicated. Analysis by SDS-PAGE confirmed expression offusion proteins.

Total cell free extracts were loaded onto an FPLC equipped with anamylose column, and the MBP-AXMI fusion proteins were purified byaffinity chromatography. Bound fusion protein was eluted from the resinwith 10 mM maltose solution. Purified fusion proteins were then cleavedwith either Factor Xa or trypsin to remove the amino terminal MBP tagfrom the AXMIz protein. Cleavage and solubility of the proteins wasdetermined by SDS-PAGE.

Example 4 Activity of Proteins Expressed from Axmiz Genes in Bioassays

Bioassay of the expressed Axmiz genes resulted in observance of thefollowing activities on insect pests:

TABLE 3 Activity of Expressed Proteins in Bioassay Plasmids Gene BCW CPBDBM ECB FAW pAX5095 Axmi221z Slight Stunt, full length No MortalitypAX7611 Axmi221z Severe stunt, full length >75% Mortality pAX5092Axmi221z Severe stunt, Severe stunt, trun2 >75% >75% Mortality MortalitypAX5094 Axmi221z Stunt Slight Stunt, trun2 No Mortality pAX7610 Axmi221zSevere stunt, trun2 >75% Mortality pAX5097 Axmi222z full length pAX7613Axmi222z Slight Stunt, Severe stunt, Severe stunt, full lengthNo >75% >75% Mortality Mortality Mortality pAX5093 Axmi222z trun2pAX5096 Axmi222z trun2 pAX7612 Axmi222z Severe stunt, Severe stunt,Stunt, No trun2 >75% >75% Mortality Mortality Mortality pAX6887 axmi223zStunt, No Severe stunt Severe stunt, Severe stunt, Stunt, No full lengthMortality >75% >75% Mortality Mortality Mortality pAX6888 axmi223zStunt, No Severe stunt Severe stunt, Severe stunt, Stunt, No alt startMortality >75% >75% Mortality Mortality Mortality pAX7634 Axmi224zSevere stunt, Strong Stunt, alt start >75% No Mortality MortalitypAX6890 axmi224z Slight Stunt, Severe stunt, Severe stunt, Stunt, No altstart <<5 No >75% >75% Mortality Mortality % Mortality MortalityMortality 5% Mortality pAX6891 axmi225z Slight Stunt, Severe stunt,Severe stunt, Stunt, No trun No >75% >75% Mortality Mortality MortalityMortality

TABLE 4 Activity of Expressed Proteins in Bioassay Plasmid Gene Hv HzSCB SWCB VBC pAX5095 Axmi221z Severe Severe stunt, full length stunt,<25% >75% Mortality Mortality pAX7611 Axmi221z Strong Stunt, StrongStunt, full length No Mortality <25% Mortality pAX5092 Axmi221z StrongStunt, Severe stunt, Severe Severe stunt, trun2 <25% >75% stunt, >75%Mortality Mortality >75% Mortality Mortality pAX5094 Axmi221z StrongStunt, Severe Severe stunt, trun2 No Mortality stunt, <25% >75%Mortality Mortality pAX7610 Axmi221z Strong Stunt, trun2 <25% MortalitypAX5097 Axmi222z Severe full length stunt, >75% Mortality pAX7613Axmi222z Severe stunt, Severe stunt, Severe stunt, fulllength >75% >75% >75% Mortality Mortality Mortality pAX5093 Axmi222zSevere stunt, Severe stunt, Severe Severe stunt, trun2 >75% >75%stunt, >75% Mortality Mortality >75% Mortality Mortality pAX5096Axmi222z Severe stunt, Severe stunt, trun2 >75% >75% Mortality MortalitypAX7612 Axmi222z Severe stunt, Severe trun2 >75% stunt, Mortality >75%Mortality pAX6887 axmi223z Stunt, No Stunt, No Severe stunt, SevereStrong Stunt, full length Mortality Mortality >75% stunt, No MortalityMortality >75% Mortality pAX6888 axmi223z Severe stunt, Strong Severestunt, Severe Strong Stunt, alt start <25% Stunt, >75% stunt, NoMortality Mortality <75% Mortality >75% Mortality Mortality pAX7634Axmi224 Severe stunt, Severe Severe stunt, Strong Severe stunt, altstart >75% stunt, <25% Stunt, No >75% Mortality >75% Mortality MortalityMortality Mortality pAX6890 axmi224z Severe stunt, Severe Severe stunt,Severe Severe stunt, alt start >75% stunt, >75% stunt, No >75%Mortality >75% Mortality Mortality Mortality Mortality pAX6891 axmi225zSevere stunt, Severe Severe stunt, Severe Severe stunt, trun >75%stunt, >75% stunt, >75% Mortality >75% Mortality >75% MortalityMortality Mortality BCW: Black cutworm CPB: Colorado Potato Beetle DBM:Diamond Back Moth ECB: European Cornborer FAW: Fall armyworm Hv:Helitothis virescens Hz: Heliothis zea SCB: Southern cornborer SWCB:Southwestern Cornborer VBC: Velvet Bean Caterpillar

Example 5 Vectoring of Genes for Plant Expression

The coding regions of the invention are connected with appropriatepromoter and terminator sequences for expression in plants. Suchsequences are well known in the art and may include the rice actinpromoter or maize ubiquitin promoter for expression in monocots, theArabidopsis UBQ3 promoter or CaMV 35S promoter for expression in dicots,and the nos or PinII terminators. Techniques for producing andconfirming promoter—gene—terminator constructs also are well known inthe art.

In one aspect of the invention, synthetic DNA sequences are designed andgenerated. These synthetic sequences have altered nucleotide sequencerelative to the parent sequence, but encode proteins that areessentially identical to the parent sequence. See, for example, Table 4.

TABLE 4 Synthetic genes encoding AXMIz proteins Nucleotide Syntheticsequence SEQ ID NO: axmi221zv02.02 6 axmi222zv02.02 7 axmi223zv03.02 8axmi224zv03.02 9 axmi225zv02.02 10 axmi221zv02.03 11 axmi222zv02.03 12axmi223zv03.03 13 axmi224zv03.03 14 axmi225zv02.03 15 axmi221zv02.04 16axmi222zv02.04 17 axmi223zv03.04 18 axmi224zv03.04 19 axmi225zv02.04 20

In another aspect of the invention, modified versions of the syntheticgenes are designed such that the resulting peptide is targeted to aplant organelle, such as the endoplasmic reticulum or the apoplast.Peptide sequences known to result in targeting of fusion proteins toplant organelles are known in the art. For example, the N-terminalregion of the acid phosphatase gene from the White Lupin Lupinus albus(GENBANK® ID GI: 14276838, Miller et al. (2001) Plant Physiology 127:594-606) is known in the art to result in endoplasmic reticulumtargeting of heterologous proteins. If the resulting fusion protein alsocontains an endoplasmic reticulum retention sequence comprising thepeptide N-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e., the“KDEL” motif, SEQ ID NO:33) at the C-terminus, the fusion protein willbe targeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Thus, this gene encodes a fusion protein that contains the N-terminalthirty-one amino acids of the acid phosphatase gene from the White LupinLupinus albus (GENBANK® ID GI: 14276838, Miller et al., 2001, supra)fused to the N-terminus of the amino acid sequence of the invention, aswell as the KDEL sequence at the C-terminus. Thus, the resulting proteinis predicted to be targeted the plant endoplasmic reticulum uponexpression in a plant cell.

The plant expression cassettes described above are combined with anappropriate plant selectable marker to aid in the selection oftransformed cells and tissues, and ligated into plant transformationvectors. These may include binary vectors from Agrobacterium-mediatedtransformation or simple plasmid vectors for aerosol or biolistictransformation.

Example 6 Transformation of Maize Cells with the Pesticidal ProteinGenes Described Herein

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine;100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and saltsother than DN62A5S are suitable and are known in the art. Embryos areincubated overnight at 25° C. in the dark. However, it is not necessaryper se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to the genes of the invention in plant cells areaccelerated into plant tissue using an aerosol beam accelerator, usingconditions essentially as described in PCT Publication No. WO/0138514.After beaming, embryos are incubated for about 30 min on osmotic media,and placed onto incubation media overnight at 25° C. in the dark. Toavoid unduly damaging beamed explants, they are incubated for at least24 hours prior to transfer to recovery media. Embryos are then spreadonto recovery period media, for about 5 days, 25° C. in the dark, thentransferred to a selection media. Explants are incubated in selectionmedia for up to eight weeks, depending on the nature and characteristicsof the particular selection utilized. After the selection period, theresulting callus is transferred to embryo maturation media, until theformation of mature somatic embryos is observed. The resulting maturesomatic embryos are then placed under low light, and the process ofregeneration is initiated by methods known in the art. The resultingshoots are allowed to root on rooting media, and the resulting plantsare transferred to nursery pots and propagated as transgenic plants.

Materials

DN62A5S Media Components Per Liter Source Chu's N6 Basal Salt Mixture3.98 g/L Phytotechnology Labs (Prod. No. C 416) Chu's N6 VitaminSolution 1 mL/L Phytotechnology Labs (Prod. No. C 149) (of 1000x Stock)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casamino acids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. D-7299)1 mL/L (of Sigma 1 mg/mL Stock)

The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite(Sigma) is added at a concentration up to 3 g/L, and the media isautoclaved. After cooling to 50° C., 2 ml/L of a 5 mg/ml stock solutionof silver nitrate (Phytotechnology Labs) is added.

Example 7 Transformation of Genes of the Invention in Plant Cells byAgrobacterium-Mediated Transformation

Ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, and incubated overnight at 25°C. in the dark. However, it is not necessary per se to incubate theembryos overnight. Embryos are contacted with an Agrobacterium straincontaining the appropriate vectors for Ti plasmid mediated transfer forabout 5-10 min, and then plated onto co-cultivation media for about 3days (25° C. in the dark). After co-cultivation, explants aretransferred to recovery period media for about five days (at 25° C. inthe dark). Explants are incubated in selection media for up to eightweeks, depending on the nature and characteristics of the particularselection utilized. After the selection period, the resulting callus istransferred to embryo maturation media, until the formation of maturesomatic embryos is observed. The resulting mature somatic embryos arethen placed under low light, and the process of regeneration isinitiated as known in the art.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A recombinant nucleic acid molecule comprisinga nucleotide sequence encoding an amino acid sequence having pesticidalactivity against a lepidopteran or a coleopteran pest, wherein saidnucleotide sequence is selected from the group consisting of: a) thenucleotide sequence set forth in any of SEQ ID NO:2, 7, 12, and 17; b) anucleotide sequence that encodes a polypeptide comprising the amino acidsequence of SEQ ID NO:24, 25, or 26; c) a nucleotide sequence thatencodes a polypeptide comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of any of SEQ ID NO:24,25, and 26; and wherein said nucleotide sequence is operably linked to aheterologous promoter.
 2. The recombinant nucleic acid molecule of claim1, wherein said nucleotide sequence is a synthetic sequence that hasbeen designed for expression in a plant.
 3. The recombinant nucleic acidmolecule of claim 1, wherein said promoter is capable of directingexpression of said nucleotide sequence in a plant cell.
 4. A vectorcomprising the recombinant nucleic acid molecule of claim
 1. 5. Thevector of claim 4, further comprising a nucleic acid molecule encoding aheterologous polypeptide.
 6. A host cell that contains the vector ofclaim
 4. 7. The host cell of claim 6 that is a bacterial host cell. 8.The host cell of claim 6 that is a plant cell.
 9. A transgenic plantcomprising the host cell of claim
 8. 10. The transgenic plant of claim9, wherein said plant is selected from the group consisting of maize,sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato,cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, andoilseed rape.
 11. A transgenic seed comprising the nucleic acid moleculeof claim
 1. 12. A recombinant polypeptide with pesticidal activityagainst a lepidopteran or a coleopteran pest, selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence ofSEQ ID NO:27 or 28; and b) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:27 or
 28. 13. The polypeptide of claim 12 furthercomprising heterologous amino acid sequences.
 14. A compositioncomprising the polypeptide of claim
 12. 15. The composition of claim 14,wherein said composition is selected from the group consisting of apowder, dust, pellet, granule, spray, emulsion, colloid, and solution.16. The composition of claim 14, wherein said composition is prepared bydesiccation, lyophilization, homogenization, extraction, filtration,centrifugation, sedimentation, or concentration of a culture ofbacterial cells.
 17. The composition of claim 14, comprising from about1% to about 99% by weight of said polypeptide.
 18. A method forcontrolling a lepidopteran, hemipteran, coleopteran, nematode, ordipteran pest population comprising contacting said population with apesticidally-effective amount of a polypeptide of claim
 12. 19. A methodfor killing a lepidopteran, hemipteran, coleopteran, nematode, ordipteran pest, comprising contacting said pest with, or feeding to saidpest, a pesticidally-effective amount of a polypeptide of claim
 12. 20.A method for producing a polypeptide with pesticidal activity,comprising culturing the host cell of claim 6 under conditions in whichthe nucleic acid molecule encoding the polypeptide is expressed.
 21. Aplant or a plant cell having stably incorporated into its genome a DNAconstruct comprising a nucleotide sequence that encodes a protein havingpesticidal activity against a lepidopteran or a coleopteran pest,wherein said nucleotide sequence is selected from the group consistingof: a) the nucleotide sequence set forth in any of SEQ ID NO:2, 7, 12,and 17; b) a nucleotide sequence that encodes a polypeptide comprisingthe amino acid sequence of any of SEQ ID NO:24, 25, and 26; and c) anucleotide sequence that encodes a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:24, 25, or
 26. 22. A method for protecting a plantfrom a pest, comprising expressing in a plant or cell thereof anucleotide sequence that encodes a polypeptide having pesticidalactivity against a lepidopteran or a coleopteran pest, wherein saidnucleotide sequence is selected from the group consisting of: a) thenucleotide sequence set forth in any of SEQ ID NO:2, 7, 12, and 17; b) anucleotide sequence that encodes a polypeptide comprising the amino acidsequence of SEQ ID NO:24, 25, or 26; and c) a nucleotide sequence thatencodes a polypeptide comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO:24, 25, or26.
 23. The method of claim 22, wherein said plant produces a pesticidalpolypeptide having pesticidal activity against a lepidopteran,hemipteran, coleopteran, nematode, or dipteran pest.
 24. A method forincreasing yield in a plant comprising growing in a field a plant of ora seed thereof having stably incorporated into its genome a DNAconstruct comprising a nucleotide sequence that encodes a protein havingpesticidal activity against a lepidopteran or a coleopteran pest,wherein said nucleotide sequence is selected from the group consistingof: a) the nucleotide sequence set forth in any of SEQ ID NO:2, 7, 12,and 17; b) a nucleotide sequence that encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:24, 25, or 26; and c) a nucleotidesequence that encodes a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO:24, 25, or 26; wherein said field is infested with a pest againstwhich said polypeptide has pesticidal activity, and wherein saidincrease in yield is relative to the yield of a plant that does notcomprise the nucleotide sequence of (a), (b), or (c).